P38 Hydrogen sulfide regulates cardiac function via S-sulfhydration of adenylyl cyclase

Abstract

We previously reported that hydrogen sulfide (H2S) may negatively regulate heart function via suppression of β-adrenoceptor function. In the present study, we found that NaHS (an H2S donor) significantly attenuated myocyte contractility, cAMP accumulation and adenyly cyclase (AC) activity in the cardiac myocytes stimulated by forskolin, a selective adenylyl cyclase activator. These data suggest that the regulatory effect of H2S involves suppression of AC/cAMP pathway. To study the molecular mechanisms, we transfected HEK293 cells with the cDNAs of AC2, AC5 and AC6, the main AC isoforms expressed in the heart. It was found that overexpression of these isoforms largely increased intracelluar cAMP level in HEK293 cells. This effect was abolished by pretreatment with NaHS and iodoacetamide, a thiol blocking agent, implying that the thiol group in AC is important to the vascular function of H2S. Western blotting analysis shows that NaHS induced S-sulfhydration of the thiol group of the cysteine residues in AC. Since cytosolic domains of AC are implicated in catalysis, we cloned the C1a domain from AC1 (AC1C1a, aa236-aa471) and C2 domain from AC2 (AC2C2D3, aa855-aa1090). We found that forskolin significantly stimulated cAMP production in the mixture of our recombinant soluble proteins of AC1C1a and AC2C2D3. These data confirm that these two segments alone may produce the same function of the full length of AC. Treatment with NaHS significantly attenuated the effect of forskolin on cAMP production in the mixture of these two recombinant proteins. In addition, NaHS also induced obvious S-sulfhydration in either of these proteins. We therefore propose that S-sulfhydration of the key cysteine residues (e.g. cys355 and/or Cys358) may block the binding of ATP to the catalytic site of AC and therefore inhibit its activity. In conclusion, we demonstrated for the first time that H2S may regulate cardiac function by suppression of AC/cAMP pathway through S-sulfhydration of AC.